assessing-metabolic-activity-whole-regenerating-zebrafish-ventricles

Assessing the Metabolic Activity of Whole Regenerating Zebrafish Ventricles <em>Ex Vivo</em> Using an Extracellular Flux Assay

The ability of zebrafish to regenerate their hearts throughout adulthood is partially attributed to metabolic adaptations. Although it has been ...

University of Oxford

In contrast to humans, fish can fully regenerate their hearts after cardiac injury. However, not all fish have the same regenerative potential, allowing comparative inter-species and intra-species analysis to identify the mechanisms controlling successful heart regeneration. Here we report a differential regenerative response to cardiac cryo-injury among different wild-type zebrafish strains. Correlating these data with single-cell and bulk RNA sequencing data, we identify oxidative phosphorylation (OXPHOS) as a positive regulator of long-term regenerative outcome. OXPHOS levels, driven by glycolysis through the malate-aspartate shuttle, increase as soon as cardiomyocyte proliferation decreases, and this increase is required for cardiomyocyte re-differentiation and successful long-term regeneration. Reduced upregulation of OXPHOS in Astyanax mexicanus cavefish results in the absence of a dynamic temporal sarcomere gene expression program during cardiomyocyte re-differentiation. These findings challenge the assumption that OXPHOS inhibits regeneration and reveal targetable pathways to enhance heart repair in humans after myocardial infarction.

Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration.

Axon guidance cues direct nerves in the heart during development, disease, and regeneration. These cues determine cardiac innervation patterning by regulating the balance between chemo-attraction and chemo-repulsion. However, the role of one of the most crucial ligand-receptor combinations among axon guidance molecules, the Slit chemo-active ligands and their Roundabout (Robo) transmembrane receptors, remains unknown during cardiac innervation patterning. To test if Slit-Robo signalling is important for cardiac innervation guidance, we analysed Slit and Robo mouse knock-outs. Constitutive  ventricles showed significantly reduced innervation, while  hearts showed temporary increased levels of innervation compared to wild-type littermate controls. Whereas innervation was not affected in mice, the phenotype seen in  ventricles was recapitulated in  mice. Detailed expression analysis identified expression of Slit2 ligand in the endothelium of the coronary vessels, while Slit3 was highly present in the coronary smooth muscle wall and in the innervation. Both the Robo1 and Robo2 receptors were present in the nerves and at low levels in the vessels. Knocking out  specifically in the endothelium recapitulated the defects seen in the constitutive  hearts. Ex vivo axon guidance cultures showed that attraction of axons extending from the ganglia was strongly reduced in ventricles with absence of endothelial Slit2 compared to wild-type controls. In the absence of endothelial Slit2, adult mice showed reduced response to challenging the sympathetic innervation. In conclusion, we have identified an important new chemo-active Slit2-Robo1 pathway required for correct cardiac innervation development.

Article	Year
Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration. Nature cardiovascular research\| PubMed ID: 41034455	2025
Endothelial Slit2 guides the Robo1-positive sympathetic innervation during heart development. eLife\| PubMed ID: 41406017	2025

Konstantinos Lekkos

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